1. Field of the Invention
A kit and method for using the kit for the qualitative demonstration or quantitative determination of epidermal growth factor receptor (EGFR) in formalin-fixed, paraffin-embedded tissue sections.
2. Prior Art
Growth factors are substances that induce cell proliferation, typically by binding to specific receptors on cell surfaces. One such growth factor is epidermal growth factor (EGF). EGF induces proliferation of a variety of cells in vivo, and is required for the growth of most cultured cells. The EGF receptor is a 170-180 kD membrane-spanning glycoprotein, which is detectable on a wide variety of cell types. The extracellular N-terminal domain of the receptor is highly glycosylated and binds EGF antibodies that selectively bind to epidermal growth factor receptor (EGFR). Agents that competitively bind to EGFR have been used to treat certain types of cancer. Sato et al., in U.S. Pat. No. 5,459,061, discloses the structure of a immunogenic segment of the EGFR protein that is presented on the cell surface. The inventors disclose a specific monoclonal antibody (Mab) produced by a hybridoma cell line which competes with EGF to bind to the segment of EGFR. Sato et al. discuss an ELISA competitive binding assay useful for the in vitro testing of three anti-EGFR Mab""s for their ability to inhibit the growth of a human cancer cell line. Sato et al. do not suggest or otherwise extend the application of the in vitro test of live cancer cell lines to the detection of EGFR in formalin-fixed specimen tissue.
Herlyn et al., in U.S. Pat. No. 5,470,571, disclose the use of radio labeled Mab 425 for treating gliomas that express EGF receptor. Herlyn et al. report that anti-EGFR antibodies may either stimulate or inhibit cancer cell growth and proliferation. Unfortunately, the inventors do not discuss a need for a test for examining suspect tissue for the presence of EGFR that would be predictive of the result (i.e., stimulation, inhibition or no effect) to be expected by administering any particular Mab.
Holzer et al., in U.S. Pat. No. 5,824,782, disclose the use of immunoconjugates, more specifically, anti-EGFR Mab""s or fragments thereof conjugated to a chemokine such as IL-8, the fusion protein binding to EGFR and exhibiting cytotoxic and/or chemotactic activity. Holzer et al. demonstrates the binding properties of their immunoconjugates by coating a substrate with EGFR, incubating the coated substrate with immunoconjugate then exposing the coated substrate to a monoclonal antibody bound to peroxidase. A chromogenic peroxidase substrate was added and the presence of the immunoconjugate on the substrate being determined photometrically. The test employs free EGFR coated on a substrate and is not suitable for examining tissue sections comprising biopsy material from a patient. They do not suggest using such a test for examining a tissue sample or for predicting the efficacy of anti-EGFR Mab""s for treating a particular tumor. Similarly, Wels et al., in U.S. Pat. No. 5,942,602, disclose a variety of compounds exhibiting specific binding to EGFR and disclose a method for imaging EGFR expressing cells. There is no disclosure of the use of anti-EGFR Mab""s for detecting EGFR in tissue samples as a method for selecting patients that will be responsive to anti-EGFR therapy or for predicting the therapeutic efficacy of any particular Mab-based therapy for cancer patients.
Other monoclonal antibodies having specificity for EGFR, either alone or conjugated to a cytotoxic compound, have been reported as being effective for treating certain types of cancer. Bendig et al, in U.S. Pat. No. 5,558,864, disclose therapeutic anti-EGFR Mab""s for competitively binding to EGFR. Heimbrook et al., in U.S. Pat. No. 5,690,928, disclose the use of EGF fused to a Pseudomonas species-derived endotoxin for the treatment of bladder cancer. Brown et al., in U.S. Pat. No. 5,859,018, disclose a method for treating diseases characterized by cellular hyperproliferation mediated by, inter alia, EGF.
Many tumors of mesodermal and ectodermal origin overexpress the EGF receptor. For example, the EGF receptor has been shown to be overexpressed in many gliomas, squamous cell carcinomas, breast carcinomas, melanomas, invasive bladder carcinomas and esophageal cancers. In addition, studies with primary human mammary tumors have shown a correlation between high EGF receptor expression and the presence of metastases, higher rates of proliferation, and shorter patient survival.
As mentioned above, attempts to exploit the EGF receptor system for anti-tumor therapy have generally involved the use of monoclonal antibodies against the EGF receptor. Magnani et al., in U.S. Pat. No. 6,008,203 disclose carbohydrates and carbohydrate analogs that bind to epidermal growth factor receptors. Methods of using such carbohydrates or analogs for a variety of uses related to the EGF receptor are also discussed. Methods for killing or inhibiting the growth of tumor cells with increased EGF receptor activity are disclosed. The preferred compositions comprise a sialylated lactose carbohydrate. The composition reportedly inhibits EGFR kinase activity thereby inhibiting the growth of EGFr-associated tumor cells.
Notwithstanding the reported therapeutic utility of compounds targeting EGFR for certain types of cancer, there have been relatively few assays developed for tumor-related EGFR expression in actual cancer patients. In U.S. Pat. No. 5,710,010, the contents of which are incorporated herein by reference thereto, Vogelstein et al disclose anti-mutated EGFR Mab""s for identifying cells expressing mutated EGFR and for diagnosing medical conditions associated therewith. In addition, they present the cDNA sequence of normal EGFR and mutated segments of the gene. It appears that both the overexpression of EGFR and the expression of mutated EGFR are characteristic of some tumors and may be used as markers for targeted therapies. There is, therefore, a need for a kit and method for determining the presence of EGFR on cancerous tissue removed from a patient as, for example, by biopsy, in order to evaluate the appropriateness of an in vivo therapy employing EGFR-targeting cytotoxic agents.
It is a primary object of the present invention to provide a kit, and a method for using the kit, operable for determining the presence of EGFR in tissue sections.
It is a further object of the invention to provide a kit, and a method for using the kit, operable for determining the presence of EGFR in tissue sections comprising tissue embedded in paraffin.
It is a yet a further object of the invention to provide a kit, and a method for using the kit, operable for determining the presence of EGFR in tissue sections that can be performed with minimal training and without expensive specialized equipment.
It is a yet a further object of the invention to provide a kit, and a method for using the kit, for selecting certain patients from a population of cancer patients wherein the selected patients have tumors that express EGFR and are most likely to respond to anti-EGFR therapy.
In accordance with the above objectives of the invention, there is provided a kit containing the reagents necessary for the qualitative demonstration of epidermal growth factor receptor (EGFR) in formalin-fixed, paraffin-embedded tissue sections. A two-step immunohistochemical staining procedure is employed which utilizes a monoclonal mouse antibody to EGFR. The anti-EGFR antibodies, which are bound to tissue antigens by incubation therewith, are detected using a peroxidase labeled polymer that is conjugated with secondary anti-mouse antibody antibodies. The enzymatic conversion of the subsequently applied chromogen, preferably DAB, results in formation of a visible reaction product at the site of the EGFR antigen. Following development of the chromogen, specimens may then be counterstained and coverslipped. Results are interpreted using a light microscope. This detection system should be applicable for both manual and automated staining.
The features of the invention believed to be novel are set forth with particularity in the appended claims. However the invention itself, both as to organization and method of operation, together with further objects and advantages thereof may be best be understood by reference to the following description taken in conjunction with the accompanying drawings in which: